Fusarium graminearumのTRI6 zinc finger

domainの核移行と安定性

誌名 JSM MycotoxinsISSN 02851466著者名 中嶋,佑一

前田,一行大里,修一金丸,京子小林,哲夫木村,真

発行元 日本マイコトキシン学会巻/号 66巻1号掲載ページ p. 13-15発行年月 2016年1月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

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JSM Mycotoxins 66 (1), 13- 15 (20 16) http:/ /dx.doi.org/1 0.2520/myco.66.13

JSM Mycotoxins

www.jstage.jst.go.jp/browse/myco

Nuclear localization and relative stability of the zinc finger domain of TRI6 trichothecene regulator

Keywords

Cys2His2 zinc finger protein; enhanced green fluorescence protein (EGFP); Fusarium graminearum; mycotoxin; transcription factor; trichothecene biosynthesis

We examined the localization of the full-length protein and zinc finger domain of the trichothecene regulator, TRI6. When fused to an enhanced green fluorescent protein (EGFP) gene, nuclear accumula­tion of the zinc finger domain, but not the full­length protein, was observed.

Fusarium graminearum produces trichothecene mycotoxins in infected cereal grains and poses serious threats to food safety 1l. Trichothecene production is regulated by a Cys2His2 zinc finger transcription factor, TRI6, which recognizes a consensus sequence of YNAGGCC2l. TRI6 regulates not only trichothecene (Trt)

genes, but also mevalonate pathway and other genes3l. Localization analyses with reporters such as gnhanced green fluorescent protein (EGFP) are often used to unveil the regulatory mechanism of transcription fac­tors. However, previous attempts to detect a GFP signal from the TRI6 fusion proteins were unsuccessful4l,Sl.

In our previous study, a strong TEFla promoter was connected to the Tri6::EGFP fusion gene (PrEFla_ Tri6::EGFP cassette) in a homologous integration vector and tar­geted downstream of the TrilOl locus in the genome of the Tri6 null mutant (parental strain; JCM 9873)4>.

Regardless of constitutive expression of Tri6::EGFP in the resulting transformant (strain 3#-14l) cultured in

100 ml of YS_60 medium in a 300 ml Erlenmeyer flask (weakly-inducing culture condition)4l, the fusion tran­scription factor did not provoke the biosynthesis of 15-acetyldeoxynivalenol (15-ADON)4l. Hence, we attempted to search for a strain with a much higher expression level of Tri6::EGFP from pools of transformants carrying

an ectopically integrated PrEFla_ Tri6::EGFP cassette. We first transformed a tri6- non-sense mutant (our

unpublished data) with a newly constructed ectopic

integration vector, pPrEFl a_ Tri6::EGFP, containing the Prma_ Tri6::EGFP cassette (Supplementary Fig 1). The transformants were cultured in 1 ml of YS_60 medium distributed to a 24-well plate (strongly-inducing culture condition)4l The amounts of 15-ADON produced were

evaluated by thin layer chromatography (TLC). The highest trichothecene producer of the PrEFla_ Tri6::EGFP transformant obtained by this method accumulated

15-ADON even in 100 ml of YS 60 culture medium (Fig.1A; upper panel). Transformation was also con­ducted using another ectopic integration vector, pPrEFla_ Tri6_ZF::EGFP, which contains only the zinc fin­ger domain of Tri6 (Supplementary Fig 1). One of the selected PrEFla_ Tri6_ZF::EGFP transformants accumu­lated a significant amount of mRNA when cultured in both trichothecene-non -inducing YG [0.5% (w/v) Difcor"' yeast extract, 2% (w/v) glucose] and trichothe­cene-inducing YS_60 media, as did the PrEFla_ Tri6::EGFP transformant (Supplementary Fig 2; see Supplementary Fig 3 for experimental detail).

Western blot analyses with an anti-EGFP antibody demonstrated that both fusion proteins were present in their intact forms, but with different degrees of frag­mentation depending on the medium used for cultur­ing (Fig.1A; lower panel; see Supplementary Fig 3 for experimental detail) . A previous immunoprecipitation study failed to detect the full-length TRI6 C-terminally fused to GFP in the total protein extract of the fusion gene overexpressor5l, also suggesting instability of TRI6 within F. graminearum cells. The use of a different protein extraction reagent [TRizol® reagent (Fischer Thermo Scientific) in this study versus non-denaturing buffer in the previous study5l] may account for the suc­cessful detection of TRI6:: EGFP in this study.

When cultured in trichothecene-non-inducing YG medium, a strong western blot signal corresponding to the size of the full-length TRI6:: EGFP was detected. This was accompanied by a much more intense signal of the 31.9 kDa fragment (Fig. 1A; marked by an asterisk). TRI6_ZF::EGFP was much more resistant to proteolytic cleavage, as evidenced by the overwhelming propor­tion of the intact fusion protein. Interestingly, the same 31.9 kDa fragment was detected on the blot of TRI6_ZF::EGFP (Fig.1A), suggesting the presence of a common internal cleavage site within the zinc finger domain (Fig. 1B; marked by an arrow) . The signal inten­sity decreased significantly when the transformants were cultured in the trichothecene- inducing YS_60 medium. Indeed, the intensity of the full-length TRI6:: EGFP band detected from the 100 ml YS_60 cul­ture was much less than that with YG culture without 15-ADON production (Fig. 1A). These results suggest that the quality of TRI6 structure, but not the quantity of TRI6, within fungal cells is critical for the induction of trichothecene biosynthesis (Fig. 1A).

Despite significant improvement in the production of trichothecene by TRI6:: EGFP in comparison with our

l 4 NAKAJIMA eta!.

(A)

TLC

6(i

45

YG YS_60

<-- 15-ADON

<-- TRI6::EGFP (52.4 kDa)

Western blot

<-- T!'l16_ZF :EGFP (39.6 kDa)

(B)

30

20

(kOa) "5cil~ ~ '=5.;,, ~ ~

<Y. 6' <Y. 6'

<-- EGFP (26.8 kDa)

*31.9kDa

··~ \~ ··~ \~ ~ .·. ~ .·

-<) ·~ -<) -~

~ ~

predicted nuclear localization sequence

136- RRHYRQHFKRFFCRYSECPQSAQDLQEVGTKGFAT -172

~-----TRI6 '--------

K167

TRI6::EGFP [ EGFP

TRI6 ZF: :EGFP EGFP I - ~p T ~~

predicted cleavage site

Fig. 1 Analyses of TRI6 fusion proteins.

JSM Mycotoxins

(C) BF EGFP DAPI

YG

YS_60

10 IJm

(A) Trichothecene production and fusion protein accumulation of transformants grown in trichothecene-inducing (YS_60) and non-inducing (YG) media. Fungal metabolites were extracted from 1 ml of culture medium with ethyl acetate and developed on a TLC plate (Kieselgel F254; Merck, Darmstadt, Germany). 15-ADON was visualized by the method described previously6l. For western blot analyses, 20 11g of total protein were loaded per lane and TRI6 fusion proteins were detected by using an anti-GFP antibody.

(B) Structure of TRI6 fusion proteins. The cleavage site predicted by western blot analysis is shown by a thick horizontal bar. The putative nuclear localization sequence predicted by using the NLS Mapper (http://nls-mapper.iab.keio.acjp) program, and putative ubiquitination sites (K29, K69 and K167) predicted by using the UbPred program (www.ubpred.org), are also indicated.

(C) GFP fluorescence of the hyphae. After staining the nucleus with DAPI, the hyphae were observed under an epifluorescence microscope.

previous constitutive expresser strain 3#-14l, only mar­

ginal fluorescence was observed in the nucleus under

an epifluorescence microscope (Fig. lC). In contrast,

nuclear localization of the TRI6 zinc finger domain was

evident as revealed by accumulation of marked green

fluorescence that merged with 4,6-diamidino-2-phe­

nylindole (DAPI) fluorescence (Fig.1C). Thus, the puta­

tive nuclear localization signal predicted to exist within

the zinc finger domain (Fig.1B) may function to import

TRI6 into the nucleus. Consistent with the results of the

western blot analyses, the intensity of the TRI6_ZF::EGFP

fluorescence was more prominent with the YG culture. In conclusion, the results of the current study dem­

onstrated that the instability of TRI6 fused to EGFP was

significantly abrogated by truncating the N-terminus

portion that constitutes the transcription factor activa­

tion domain. The presence of the activation domain

appears to destabilize TRI6 by stimulating proteolytic

cleavage within the zinc finger domain.

Yuichi Nakajimal, Kazuyuki Maedal, Shuichi Ohsato2, Kyoko Kanamarul, Tetsuo Kobayashil, Makoto Kimural

1Department of Biological Mechanisms and Func­tions, Graduate school of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan

2Department of Agriculture, Graduate School of Agricu lture, Meiji University, 1-1-1 Higashimita, Kawasaki, Kanagawa 214-8571, Japan

Correspondence

Yuichi Nakajima E-mail: nakajima.yuichi@a.mbox.nagoya-u.acJp

(Received December 10, 2015, accepted January 7, 2016)

Vol. 66, No. 1, 13- 15 (2016)

Acknowledgements

This work was supported by a Grant from Noda Institute for Scientific Research.

Supplementary Materials

Supplementary materials may be found in the online version of this article: Supplementary Fig. 1 Construction of ectopic integra­tion vector pPrEFla_ Tri6::EGFP and pPrEFla_ Tri6_ZF::EGFP. Supplementary Fig. 2 Northern blot analysis of the transformants. Supplementary Fig. 3 Experimental flow chart. Supplementary Fig. 4 Structure and sequence of pAn­Tef-hph.

References

1) Kimura, M., Tokai, T., Takahashi-Ando, N., Ohsato, S., Fujimura, M.: Molecular and genetic studies of Fusarium

15

trichothecene biosynthesis: pathways, genes, and evolu­tion. Biosci Biotechnol Biochem, 71, 2105-2123 (2007)

2) Hahn, T. M., Krishna, R., Proctor, R. H.: Characterization of

a transcriptional activator controlling trichothecene toxin biosynthesis. Fungal Genet Bioi, 26, 224-235 (1999)

3) Seong, K. Y., Pasquali, M., Zhou, X., Song, J., Hilburn, K., McCormick, S., Dong, Y., Xu, J. R., Kistler, H. C.: Global gene regulation by Fusarium transcription factors Tri6 and TrilO reveals adaptations for toxin biosynthesis. Mol Microbial, 72, 354-367 (2009)

4) Nakajima, Y., Tokai, T., Maeda, K., Tanaka, A., Takahashi­Ando, N., Kanamaru, K., Kobayashi, T., Kimura, M.: A set of heterologous promoters useful for investigating gene functions in Fusarium graminearum. JSM Mycotoxins, 64, 147-152 (2014)

5) Hou, R., Jiang, C., Zheng, Q., Wang, C., Xu, J. R.: The AreA

transcription factor mediates the regulation of deoxyni­valenol (DON) synthesis by ammonium and cyclic ade­nosine monophosphate (cAMP) signalling in Fusarium

graminearum. Mol Plant Pathol, 16, 987-999 (2015)

6) Takitani, S., Asabe, Y., Kato, T., Suzuki, M., Ueno, Y.: Spec­trodensitometric determination of trichothecene myco­toxins with 4-{p-nitrobenzyl)pyridine on silica gel thin­layer chromatograms. J Chromatogr, 172, 335-342 (1979)

98 JSM Mycotoxins

FlIsarillm graminearllmのTRI6zinc finger domαinの核移行と安定性

中嶋佑-1，前田一行1，大里修一2，金丸京子l，小林哲夫人木村真1

1名古屋大学大学院生命農学研究科(干464-8601 愛知県名古屋市千種区不老町)

2明治大学大学院農学研究科(干214・8571 神奈川県川崎市多摩区東三田1-1-1)

トリコテセン生合成制御因子TRI6の全長および、zincfinger domain の局在解析を試みた.緑色蛍光タンパク質EGFP

との融合実験でTRI6zinc finger domain は核への蓄積が確認されたがTRI6全長は局在解析が困難であった

キーワード:転写因子;トリコテセン生合成，マイコトキシン(かぴ毒);緑色蛍光タンパク質;Cys2His2型Znフインガータンパク質;

Fusαriumgr，αmineαrum

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